Dephosphorylation of Fbx15 is required for nuclear clearance of SsnF

During stress conditions Fbx15 becomes rapidly dephosphorylated, presumably by the essential protein phosphatase 2A catalytic subunit GlcA/BimG (Figure 37). GlcA belongs to the serine/threonine phosphatases and shares homology with the yeast protein phospha-tase 1 (PP1) catalytic subunit Glc7 (Winkelströter et al., 2015). GLC7 is essential for yeast as well, but conditional mutant alleles of GLC7 could be connected to defects in adaptive functions like temperature tolerance, glucose repression, amino acid starvation, cell morphology and DNA damage repair, which are reminiscent to the growth defects of the ∆fbx15 mutant on the respective conditions (Andrews and Stark, 2000; Hu et al., 2012; Stark, 1996; Zeska Kress et al., 2012). The dephosphorylation of Fbx15 on Ser468/Ser469 finally led to cytoplasmic accumulation of SsnF, whereas nuclear SsnF was cleared.

104 Discussion

Figure 37: Model for Fbx15 function and localization during vegetative growth and stress response in A. fumigatus. During vegetative growth a significant part of Fbx15 is phosphorylated, potentially by the cyclin-dependent kinase NimX. Most of this phosphorylated Fbx15 is transported together with the transcriptional repressor adaptor SsnF into the nucleus. In the nucleus SsnF forms a functional co-repressor complex with a tetramer of RcoA, which interacts with DNA binding proteins for the repression of stress and secondary metabolite target genes. The small fraction of cytoplasmic as well as the larger fraction of nuclear phosphorylated Fbx15 can be integrated into active SCF^Fbx15 E3 ubiquitin ligases. Grey arrows reflect the situation of phosphorylated Fbx15 under vegetative growth conditions. Fbx15 interacts with the phosphatase GlcA and becomes dephosphorylated and produced in higher levels under stress conditions (red arrows).

Dephosphorylated Fbx15 can be integrated into less active nuclear SCF complexes.

Dephosphorylated Fbx15 results in less SsnF in the nucleus, which can be caused by a reduced import or an enhanced export of the co-repressor from the nucleus or a combination of both. Reduced nuclear SsnF results in the de-repression of stress response and secondary metabolite genes like gliP.

One possibility for the Fbx15-dependent nuclear clearance of SsnF is of course the ubiquitin mediated proteasomal degradation of SsnF. However, SsnF was not found to be ubiquitinated and the overall cellular pool of SsnF appeared to be stable independent of external stress or Fbx15. Therefore, ubiquitin-dependent degradation of SsnF seems un-likely. However, previous studies have shown that some E3 ubiquitin ligases can directly

interact with the regulatory particle of the proteasome and thus are able to transfer target proteins to the proteasome for degradation (Xie and Varshavsky, 2002). A similar scenar-io could be responsible for selective nuclear degradatscenar-ion of SsnF, where dephosphory-lated Fbx15 incorporates into inactive SCF-core complexes, which have the potential to carry specific target substrates such as SsnF directly to the proteasome. This is also sup-ported by the fact that dephosphorylated Fbx15 interacts with SkpA primarily in the nu-cleus by forming inactive SCF^Fbx15 core complexes.

Another possibility for the reduction of nuclear SsnF upon external stress might be a reduced nuclear import or an Fbx15-dependent nuclear export mechanism. The data from this work indicates a combination of both mechanisms, since SsnF seemed to be stuck at nuclear pore complexes upon stress, in an Fbx15-dependent manner. In addition, an Fbx15 dependent SsnF-export mechanism might facilitate the rapid nuclear clearance of SsnF, while the cellular pool of SsnF remains stable.

Both phosphorylated and dephosphorylated Fbx15 are able to interact with SsnF. The difference between both phosphorylation states of Fbx15 is the cellular interaction site.

Under normal growth conditions the primarily phosphorylated version of Fbx15 seems to interact with SsnF predominantly in the cytoplasm, indicating a cargo function for the nuclear import of SsnF, which is released in the nucleus. Stoichiometric abundances of Fbx15 and SsnF were not balanced after all, especially under non-stress conditions, fur-ther arguing against stable Fbx15-SsnF complexes. In addition a potential ubiquitinating function of Fbx15 towards the nuclear pore complex (NPC) subunit Nic96, which was co-purified during our TAP-tag pull-downs, might be a reasonable function for SCF^Fbx15-ligase complexes to promote nuclear transport control. Although the localization of SsnF-GFP in ∆fbx15 background and the localization of Nic96-GFP shared some simi-larities, no Fbx15-specific or stress-dependent changes in the ubiquitination pattern for Nic96 could be observed. However, the nuclear pore complex is a massive multi-protein complex composed of 30 different NPC-proteins, which are arranged in multiples and finally reach a molecular mass between 66 and 125 MDa (Dokudovskaya et al., 2007;

Grossman et al., 2012). In 2012 Hayakawa et al. showed that approximately half of the NPC-proteins in yeasts are ubiquitinated, but not necessarily targeted for proteasomal degradation (Bailey and Elkan, 1994; Hayakawa et al., 2012). It might be possible that Fbx15 plays a role in NPC-protein ubiquitination, which targets NPC-proteins in close proximity to Npc96 and thereby promotes a more general nuclear transport control.

106 Discussion

Under stress conditions Fbx15 expression is induced. In addition stress leads to the dephosphorylation of Fbx15, which then seems to interact with SsnF primarily in the nu-cleus, finally resulting in reduced SsnF amounts in the nucleus. This could imply a nucle-ar export function, where Fbx15 acts as a cnucle-argo receptor for SsnF (Figure 37).

1.6 The phosphorylation state of Fbx15 might determine its nuclear/cytoplasmic localization

Fbx15 interacts not only with the SsnF adaptor for transcriptional repressors but also with the adaptor protein SkpA, which bridges Fbx15 into SCF E3 ubiquitin ligase complexes.

Similar to the interaction with SsnF the interaction between Fbx15 and SkpA was not disturbed due to the dephosphorylation of Fbx15 upon oxidative stress, but rather shifted from the cytoplasm to the nucleus. The identified phosphorylation site of Fbx15, Ser469 with Ser468 as an alternative, putatively redundant phospho-acceptor, is located between both nuclear localization sites (NLS). So, phosphorylation/dephosphorylation events on Fbx15 Ser468/469 might determine nuclear import/export of Fbx15 by rearranging the NLSs rather than protein-binding specificity. The localization pattern of Fbx15 seems to have a major influence on the nuclear localization of SsnF, but disturbed localization pat-terns of SsnF in constantly unphosphorylated fbx15 mutants resulted only in an interme-diate phenotype. This suggests a second mechanism for Fbx15, which is required for complete stress tolerance in A. fumigatus. This is presumably based on the canonical function of Fbx15 as part of an SCF E3 ligase complex. The formation of active SCF^Fbx15 ligase complexes was promoted in fbx15 mutants, which mimic a constant phosphoryla-tion, which further suggests an ubiquitinating function of Fbx15-carrying SCF ligases during non-stress conditions. Since Fbx15 abundance under non-stress conditions is very low and overall ubiquitin-patterns of the cellular pool of proteins were not significantly changed between wild type and ∆fbx15 mutants, the putative target(s) of SCF^Fbx15 are presumably highly specific.

2 The development – virulence connection in fungi